Coated substrate for biological reaction systems

ABSTRACT

An apparatus for biological reactions is provided. The apparatus includes a substrate and a plurality of reaction sites within the substrate. A surface of the substrate is configured to have a first hydrophilicity and each surface of the plurality of reaction sites is configured to have a second hydrophilicity to load a substantial number of reaction sites with a sample volume. The sample volume of each loaded reaction site is substantially confined to its respective reaction site. The sample volume is configured to undergo a biological reaction within the reaction site.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority of U.S. ProvisionalPatent Application No. 61/612,005, filed on Mar. 16, 2012, U.S.Provisional Patent Application No. 61/612,087, filed on Mar. 16, 2012,U.S. Provisional Patent Application No. 61/723,759, filed on Nov. 7,2012, U.S. Provisional Patent Application No. 61/612,008, filed Mar. 16,2012, U.S. Provisional Patent Application No. 61/723,658, filed Nov. 7,2012, and U.S. Provisional Patent Application No. 61/723,738, filed onNov. 7, 2012, all of which are also incorporated herein in theirentirety by reference.

BACKGROUND

The present disclosure relates to a method of treating a surface of asubstrate used in a biological reaction system, and more particularly,to a method of chemically treating a surface of a substrate used in abiological reaction system to prevent biological molecules from adheringto the surface.

Polymerase Chain Reaction (PCR) is a method of amplifying a target DNAsequence. Previously, PCR has been generally performed in 96- or384-well microplates. If higher throughputs are desired, conventionalPCR methods in microplates are not cost effective or efficient. Further,in increasing throughput, reducing the PCR reaction volumes may lowerthe consumption of reagents, leading to a decrease in amplificationtimes from the reduced thermal mass of the reaction volumes. Thisstrategy may be implemented in an array format (m×n), resulting in alarge number of smaller reaction volumes. Furthermore, using an arrayallows for a scalable high throughput analysis with increasedquantification sensitivity, dynamic range, and specificity.

Arrays have also been used to perform Digital Polymerase Chain Reaction(dPCR). Results from dPCR can be used to detect and quantify theconcentration of rare alleles, to provide absolute quantitation ofnucleic acid samples, and to measure low fold-changes in nucleic acidconcentration. Generally, increasing the number of replicates increasesthe accuracy and reproducibility of dPCR results.

The array format in most quantitative polymerase chain reaction (qPCR)platforms is designed for sample-by-assay experiments, in which PCRresults need to be addressable for post-run analysis. For dPCR, however,the specific position or well of each PCR result may be immaterial andonly the number of positive and negative replicates per sample may beanalyzed.

The read-out of dPCR, that is, the number of positive reactions and thenumber of negative reactions, is linearly proportional to the templateconcentration, while the read-out of qPCR (signal vs. cycle) isproportional to the log of the template concentration. Thus, for dPCR,it is desirable to minimize sample volumes.

However, continuing to decrease reaction volumes may lead to challengesrelated to confidence in loading the array with sample volumes andmaintaining the physical isolation of the sample volumes, for example.In other words, it is important to load the sample volume into as manywells or through-holes as possible and to reduce the cross-talk betweenthe wells or through-holes.

SUMMARY

In one exemplary embodiment, an apparatus for biological reactions isprovided. The apparatus includes a substrate and a plurality of reactionsites within the substrate. A surface of the substrate is configured tohave a first hydrophilicity and each surface of the plurality ofreaction sites is configured to have a second hydrophilicity to load asubstantial number of reaction sites with a sample volume. The samplevolume of each loaded reaction site is substantially confined to itsrespective reaction site. The sample volume is configured to undergo abiological reaction within the reaction site.

DESCRIPTION OF THE FIGURES

FIG. 1 is an exemplary coated substrate according to various embodimentsof the present teachings;

FIG. 2 illustrates a receding and advancing contact angles according tovarious embodiments of the present teachings.

FIG. 3 illustrates loading of reaction sites by a sample loaderaccording to various embodiments of the present teachings;

FIG. 4 is a flowchart illustrating an exemplary method according tovarious embodiments of the present teachings;

FIG. 5 illustrates a step in coating a substrate surface of a substrateaccording to various embodiments of the present teachings;

FIG. 6 illustrates a step in coating a vertical surface of a substrateaccording to various embodiments of the present teachings;

FIG. 7 illustrates another step in coating the substrate surface of asubstrate according to various embodiments of the present teachings;

FIG. 8 illustrates another step in coating a vertical surface of asubstrate according to various embodiments of the present teachings; and

FIG. 9 illustrates a block diagram of an exemplary polymerase chainreaction instrument that may use substrates coated according to variousembodiments of the present teachings.

DETAILED DESCRIPTION

To provide a more thorough understanding of the present invention, thefollowing description sets forth numerous specific details, such asspecific configurations, parameters, examples, and the like. It shouldbe recognized, however, that such description is not intended as alimitation on the scope of the present invention, but is intended toprovide a better description of the exemplary embodiments.

Performing several biological reactions simultaneously may require asubstrate with a plurality of reaction sites, each with a loaded sample.It should be recognized that reaction sites may be, but are not limitedto, through-holes, indentations, or wells, for example. Further, toincrease the number of reactions per experiment, the density of reactionsites on the substrate may be increased while also reducing the samplearea size. For example, on a 15 mm×15 mm substrate, 10,000 reactionsites may be included. However, if 30,000 reaction sites are included inthe 15 mm×15 mm substrate, the sample area may be smaller as well asincreasing the density of reaction sites on the substrate.

According to various embodiments described herein, an apparatus that maybe sufficiently loaded with a sample volume is provided. Surfacecharacteristics such as the hydrophobicity and/or hydrophilicity ofspecific areas of the substrate may facilitate loading of liquid samplesinto the reaction sites. The level of hydrophobicity/hydrophilicity maybe based on various factors that influence the ease and the efficiencyof loading a plurality of reaction sites in a substrate as describedabove.

For example, one factor that affects loading of reactions sites is thephysical geometry of the reaction sites and/or substrate may facilitateloading the sample. For example, the aspect ratio between reactionsites, based on reaction site depth (chip thickness) and diameter of thereaction sites, may be a factor in determining the characteristicsneeded for sufficient loading of the reaction sites. The ratio ofreaction site depth (chip thickness) to diameter is referred to asaspect ratio. For example, the aspect ratio is 1 if the reaction sitedepth is equal to the reaction site diameter. In another example, theaspect ratio of 10 when reaction site depth is 10× larger than reactionsite diameter. The diameter of the reaction site affects the capillaryforces, which enable/facilitate reaction sites loading with the liquidreaction media. In some embodiments, the smaller the diameter, thelarger capillary force and better/easier loading of the reaction sites.

Another factor that affects the loading of reaction sites with a samplevolume according to various embodiments is the desired loadingefficiency and/or consistency. Desired efficiency is more than or equalto 90% of the reaction sites loaded. In other embodiments, desireefficiency may be 90% of the reaction sites loaded and a reactionsite-to-reaction site variation of at most 10%.

Another factor that affects the loading of reaction sites with a samplevolume according to various embodiments is the compatibility ofmaterials with a desired reaction within the reaction sites. In someembodiments, the desired reaction may be an amplification reaction. Morespecifically, the amplification reaction may be a polymerase chainreaction (PCR). The portion of an apparatus, according to variousembodiments, which contacts the sample volume, enzymes, or reagents, forexample, involved in the reaction should not chemically interact withthe sample volume, enzyme, or reagents. For example, materials that arein contact with the reaction should not leach ions into to the reactionsite which may interfere with the reaction.

Yet another factor that affects the loading of reaction sites accordingto various embodiments is the desired confinement of the sample volumeonce it is loaded into the reaction site. In other words, sufficientforces to prevent leakage of the sample volume from each reaction site,to prevent spilling over from one reaction site to another, and toprevent any pooling of sample volume outside the reaction sites isdesired.

Taking into account the above-mentioned factors, an apparatus that canmeet these goals may be designed and used to perform reactions in aplurality of reaction sites including small volumes.

To create the sufficient hydrophobic/hydrophilic characteristics of asurface, the surfaces may be coated with another material. According tovarious embodiments of the present teachings, a coating for a portion ofa substrate surface that has hydrophilic characteristics and a coatingfor a portion of a surface of a reaction site that has hydrophiliccharacteristics. Although both surfaces have hydrophiliccharacteristics, the reaction sites may be loaded by capillary action,for example. According to various embodiments, the hydrophilic coatingmay be the same coating. According to various embodiments, the coatingmay be applied by vapor deposition.

On the other hand, according to various embodiments of the presentteachings, a coating for a portion of a substrate surface that hashydrophobic characteristics and a coating for a portion of a surface ofa reaction site that has hydrophobic characteristics. Although bothsurfaces have hydrophobic characteristics, the reaction sites may beloaded by capillary action, for example. According to variousembodiments, the hydrophobic coating may be the same coating. Accordingto various embodiments, the coating may be applied by vapor deposition.

Further, according to other embodiments described herein, a coatingmethod for a substrate that includes coating a portion of a substratesurface to have a hydrophobic characteristic and a portion of thesurface of the substrate to have a hydrophilic characteristic mayfacilitate. Further, according to some embodiments, the surfaces of thesample area are coated to be hydrophilic and the other surfaces of thesubstrate are coated to be hydrophobic. In this way, thehydrophobicity/hydrophilicity facilitate loading of liquid samples intothe reaction sites. The reaction sites may be loaded by capillaryaction, for example.

Furthermore, according to various embodiments, havinghydrophobic/hydrophilic surfaces may reduce cross contamination ofliquid samples between reaction sites. The hydrophobic areas may helpkeep each liquid sample in its respective sample area within a reactionsite. According to various embodiments, a continuous or complete coatingmay also prevent or reduce leaching of ions from the substrate that mayinterfere with the reactions.

A coating, according to various embodiments, has increased physical andchemical stability than other coatings, and is biocompatible. This mayprevent or reduce adsorption and subsequent inhibition of the activebio-chemicals and materials used in biological reactions. These mayinclude enzymes, probes, and DNA, for example. Especially for digitalPCR applications, it is important that reaction chemicals and componentsadsorption to undesired surfaces is minimized.

Substrate

According to various embodiments described herein, substrate materialsmay be silicon, silicon oxide, glass, metals, ceramic, or plastic, forexample. The glass may be photosensitive glass. However, one skilled inthe art would recognize that any material that is biocompatible and doesnot interfere with fluorescence detection may be coated according tovarious embodiments of the present teachings.

As mentioned above, reduction in reaction volumes may allow for a higherdensity of reaction volumes so that more reactions can be performedwithin a given area. For example, an array comprised of 300 μm diameterthrough-holes in a substrate may contain about 30 nL of reaction volume.By reducing the size of each through-hole in an array to 60-70 μm indiameter, for example, each reaction volume may be 100 pL. According tovarious embodiments described herein, reaction volumes may range fromabout 1 pL to 30 nL. In some embodiments, an array of reaction sites maybe comprised of a variety of different volume reaction areas so thatdynamic range is increased.

FIG. 1 illustrates chip 100 including a substrate 100 with an array ofreaction sites 104 according to various embodiments described herein.Chip 100 may be referred to as an article, device, consumable, array,slide or platen, for example.

Chip 100 comprises a substrate 100. Substrate 102 may be variousmaterials including, but not limited to, metal, glass, ceramic, siliconand silicon oxide, for example.

Chip 100 further includes a plurality of reaction sites 104. Theplurality of reaction sites 104 may be wells, cavities, orthrough-holes, for example. Each sample area may also have a variety ofcross-sectional geometries, such as round, triangular, or hexagonal, forexample. Having other geometries may allow for more closely packedreaction sites to further increase the number of reactions in a givenarea. Further, geometries of the reaction sites may also facilitateloading of liquid samples to the reaction sites.

The cross-sectional view of chip 100 in FIG. 1 illustrates a pluralityof through holes 104. Each through-hole 104 extends from an opening in afirst surface of substrate 102 to an opening in second surface ofsubstrate 102, each through holes 104 being configured to providesufficient surface tension by capillary action to hold respective liquidsamples containing a biological sample to be processed or examined. Chip100 may have a general form or construction as disclosed in any of U.S.Pat. Nos. 6,306,578; 7,332,271; 7,604,983; 7,682,565; 6,387,331; or6,893,877, which are herein incorporated by reference in their entiretyas if fully set forth herein.

According to various embodiments, a through-hole 104 may have a volumeof about 1.3 nanoliters. Alternatively, the volume of each through-holemay be less than 1.3 nanoliters, for example, by decreasing the diameterof through-holes 104 and/or the thickness of substrate 102. For example,each through-hole 104 may have a volume that is less than or equal to 1nanoliter, less than or equal to 100 picoliters, less than or equal to30 picoliters, or less than or equal to 10 picoliters. In otherembodiments, the volume some or all of the through-hole 104 is in arange of 1 to 20 nanoliters.

In certain embodiments, a density of through-holes 104 may be at least50 through-holes per square millimeter. In other embodiments, there maybe higher densities of through-holes. For example, a density ofthrough-holes 104 within chip 100 may be greater than or equal to 150through-holes per square millimeter, greater than or equal to 200through-holes per square millimeter, greater than or equal to 500through-holes per square millimeter, greater than or equal to 1,000through-holes per square millimeter, greater than or equal to 10,000through-holes per square millimeter.

Other embodiments of chip 100 are further described in provisionalapplications 61/612,087, filed on Mar. 16, 2012, and 61/723,759, filedNov. 7, 2012, which are incorporated herein for all purposes.

As mentioned above, reducing the size of a sample area may lead tochallenges associated with loading the liquid sample into each samplearea. A coating applied to the substrate surface according to variousembodiments described herein may facilitate loading of liquid samples toreaction sites as well as minimize cross-talk between reaction sites.

Before coating, according to embodiments described herein, thesubstrates may be cleaned and hydrated to prepare them for thesubsequent chemical reactions. Cleaning removes any possiblecontaminations that may have occurred during transport and storage, forexample, to ensure consistency in the coating process.

It should be recognized that the methods and protocols described in thisdocument are examples according to various embodiments described herein.The protocols may be modified to work on high aspect ratio chips and/orlow aspect ratio chips. In some embodiments, sufficient loading mayoccur when the water contact angle is between 60-100 degrees. In otherembodiments, sufficient loading may occur when the water contact angleis 75-90 degrees.

Further, both bi-coats and mono-coats may achieve coating as describedby various embodiments. Moreover, coating methods according to variousembodiments may include a liquid coating process as well as vapordeposition processes, for example.

Hydrophilicity Method

As mentioned above, according to various embodiments of the presentteachings, a hydrophilic coating of the substrate surface as well as onthe surface of the reaction sites may be used to facilitate preparationof an array of reaction sites for a biological reaction. Also mentionedabove, a substrate may be referred to as a chip, and reaction sites maybe, but are not limited to, through-holes, indentations, or wells, forexample. According to various embodiments, the coating of the substratesurface and reaction sites may be the same material. In otherembodiments, the coating of the substrate surface and the reaction sitesmay be different materials. The substrate surface and the surface ofreaction sites may be coated by a vapor deposition process.

Even though both the substrate surface and the reaction site surface mayhave hydrophilic characteristics according to various embodiments,loading of the liquid sample may be based on capillary action. In otherwords, adhesion forces between the liquid sample and the walls of thereaction site will pull the liquid sample into the reaction sites. Fromthe equation below, the amount of liquid sample that may be pulled intoeach reaction site depends on the radius of the reaction site.

The height h of a liquid column is given by:

$h = \frac{2\;{\gamma cos}\;\theta}{pgr}$

where γ is the liquid-air surface tension (force/unit length), θ is thewater contact angle, ρ is the density of liquid (mass/volume), g islocal gravitational field strength (force/unit mass), and r is radius ofreaction site (length).

According to various embodiments, the hydrophobic/hydrophiliccharacteristics of the substrate surface and the reaction sites maydepend on the material the substrate and reaction sites are composed ofand/or the material of the coating of those surfaces. The efficiency ofloading of the reaction sites depends on the water contact angle of theliquid sample with the substrate surface and reaction site surfaces.

The spreading of the liquid sample from the sample loader depends on thewater contact angle of the liquid sample. The water contact angleresults from the relationship of the material properties of the sampleloader with the properties of the liquid sample. When the water contactangle is less than 90 degrees, the relationship between the liquidsample and the substrate surface is hydrophilic and the sample exhibitsa cohesive interaction with the substrate surface, which is necessaryfor capillary action to pull the sample into the through holes. Asubstrate that is too hydrophilic, for example, with a water contactangle below 50 degrees, may lead to increased pooling of excess liquidsample on the substrate surface, or inefficient loading of reactionsites, for example. Further, low contact angles may cause the liquidsample to move into some reaction sites too quickly resulting in anuneven distribution of liquid sample in the plurality of reaction sites.

Conversely when the water contact angle is over 90 degrees, therelationship between the substrate surface and the liquid sample ishydrophobic and the liquid sample will not move into the reaction sites,because the capillary force will be negative. This situation may alsolead to pooling of liquid sample on the substrate surface and preventloading of some reaction sites with liquid sample. As such, surfaces ofthe substrate and the reaction sites are designed to balance thehydrophobicity and hydrophilicity of the substrate and reaction sitessurfaces with respect to the liquid sample.

With these characteristics in mind, according to various embodiments,efficient loading may be achieved by configuring the sample loader sothat the advancing contact angle with the liquid sample is similar tothe receding contact angle with the liquid sample. With reference toFIG. 2, advancing and receding contact angles are illustrated. A waterdroplet 202 is shown on a substrate 200. If the substrate is tilted,water droplet 202 will have an advancing contact angle 206 and areceding contact angle 204.

According to various embodiments described herein, an advancing contactangle of 70-85 degrees may provide sufficient loading of the liquidsamples to the reaction sites.

The difference between the advancing and receding contact angles isknown as the hysteresis. In various embodiments, the surfacecharacteristics may be designed so that there is a hysteresis of zerodegrees. In other embodiments, the surface characteristics may bedesigned so that there is a hysteresis of less than or equal to 30degrees. In other embodiments, the surface characteristics may bedesigned so that there is a hysteresis of less than or equal to 20degrees. In other embodiments, the surface characteristics may bedesigned so that there is a hysteresis between 0-15 degrees. A chip withhigh hysteresis will exhibit variation in the loaded volume in thethrough-holes, and may be prone to sample pooling on exterior surfaces.

With reference to FIG. 3, loading of reaction sites by a sample loaderis illustrated according to various embodiments described herein. Theliquid sample 304 to be loaded into reaction sites 104 is within sampleloader 302. Sample loader 302 is laterally moved across surface 106. Asit is moved, liquid sample 304 is loaded into reaction sites 104 bycapillary action.

As mentioned above, the coating according to various embodiments may bedeposited by a vapor deposition process. An example of such a process isas follow:

-   -   1. Soak chips in 2-propanol for 30 seconds with sonication    -   2. Sonicate in 5:1:1 water:ammonium hydroxide:hydrogen peroxide        v:v (from 30% stock solutions of hydrogen peroxide and ammonium        hydroxide) at 60° C. for 10 minutes.    -   3. Sonicate in 4:1 water:nitric acid v:v (from 70% stock        solution of nitric acid) at 60° C. for 10 minutes.    -   4. Dry    -   5. Vapor phase deposition of Hexamethyldisilazane (HMDS)

It should be recognized that other suitable coatings according tovarious embodiments described herein are alkyl-trimethoxysilanes,alkyl-triethoxysilanes, alkyl-dimethoxymonomethylsilanes,alkyl-diethoxymonomethylsilanes, alkyl-monomethoxydimethylsilanes, oralkyl-monoethoxydimethylsilanes, or any other silane, siloxane, silazaneor phosphonate that is volatile under vacuum at elevated temperatures.

It should also be recognized that coating materials that create aslightly hydrophobic substrate surface and the surface of the reactionsite may also be used to load reaction sites according to variousembodiments described herein. Advancing water contact angles andreceding water contact angles of 90-100 degrees may also be sufficientto load reaction sites according to various embodiments.

Hydrophilic/Hydrophobic Method

According to various embodiments described herein, below is an exampleof a coating process for achieving the desired characteristics forsufficient loading. The exemplary method is shown in FIG. 4.

Substrate Surface of Chip First Step: Hydrophobic Silanes (C1)

The method includes step 402 of creating a hydrophobic layer onsubstrate surface 106 (FIG. 1). With reference to FIG. 5, step 402 isillustrated where a hydrocarbon layer is added to substrate surface 106by reacting the hydrated substrate surface with reactive tri-alkoxysilanes. One can fine tune 106 surface properties by choosing the properhydrocarbon chain as the fourth functional group of the reactingtri-alkoxy silane.

Surface properties of substrate surface 106 are critical for consistentsample loading into reaction sites 104 and also to prevent samplepooling/remaining on substrate surface 106 after loading in reactionsites 104. Pooling of sample may lead to cross talk between adjacentreaction sites during the bio-reaction cycles and also may lead tosubsequent false positive results. According to various embodiments,surface properties may be fine tuned with the length of hydrocarbonchain, the fourth functional group of silanes, to change surfaceproperties, for example.

In one embodiment, illustrated in FIG. 5, 106, the substrate surfaces ofthe substrate are exposed and reacted with tri-alkoxy silane (thehydrocarbon chain can be from C4 to C20) dissolved in non-polar solvent,such as heptanes. Then, prior to immersing the substrates in silanesolution, the reaction sites are filled with dilute acid solution inwater which will protect the respective surfaces from reacting with thesilanes dissolved in heptanes which are immiscible with water. Thereaction between the alkoxy-silane and the hydrated surfaces continue tooccur for given period of time. These reactions are completed byhydrolysis in acidic solutions and air. After reaction completion, thewater is removed via washing with various solvents such as acetonitrile, isopropyl alcohol and heptanes and the surfaces are preparedfor the next step in the method, according to various embodiments.

Reaction Site Surfaces First Step: Functionalized Silanes (C2)

After rendering the substrate surface hydrophobic as in FIG. 5 (step 402of FIG. 4), step 404 includes a process of coating the vertical surfacesof the reaction sites to be hydrophilic with a substantially continuousfilm. In this way, the sample volumes including bio-chemicals may bemore easily loaded into each desired reaction site.

In this example, S1 represents solvent 1 (hydrophobic) and S2 representssolvent 2 (hydrophilic). Here, S1 and S2 are immiscible solvents with S1not having access to the area wetted by S2. C1 represents alkoxy silanesdissolved in S1. C1 may react with —OH groups present on the substratesurface 106. The reaction illustrated in FIG. 5 renders substratesurface 106 hydrophobic. The presence of S2 prevents S1 and the alkoxysilane from accessing the vertical surfaces of the reaction sites 104,maintaining the hydrophilicity.

Sample loading and retention in reaction sites during reactions may becontrolled and enhanced by the aspect ratio and type of coating appliedto the chip surfaces. Furthermore, some chip substrates may leach ionsor have physical interaction with samples (bio)-chemicals, asubstantially continuous coating may be needed. A substantiallycontinuous coat is a coating that does not allow ions to leach into thereagent media.

In one example, a substantially continuous coating may be achieved withdouble functionalized silanes (alkoxy and epoxy groups), with the alkoxygroup binding to the hydrated substrate, while the epoxy group mayenable the subsequent reactions with hydrophilic polymers.

One embodiments of this step is illustrated in FIG. 6. As completed inFIG. 5, the substrate surface of the substrate has been covered withC4-C20 functionalized silane. The (Alkoxy)3-Si-glycidyl or aminofunctionalized silane might also react with unreacted —OH groups on thesubstrate surface 106.

More specifically, in the example illustrated in FIG. 6, C2 represents(Alkoxy)₃ glycidyl of amino functionalized silanes, which are dissolvedin S1 and may react with —OH groups present on the surfaces of thereaction sites 104. In other words, the reaction site hydrated walls mayreact with a solution of tri-alkoxy silane functionalized with glycidylgroup dissolved in heptanes.

The reaction the surfaces of the reaction sites 104 renders somehydrophilicity to the surfaces of the reaction sites 104 and providesthe functional groups for the next steps of the process to cover thehorizontal walls with a protective, continuous layer. In other words,the glycidyl group may attach to the reaction site walls via the silanelinked to the hydrated surface and may allow further chemistry tofine-tune the coating of the reaction sites to enable applications, suchas dPCR, for example.

Reaction Site Surfaces Second Step: Attachment of FunctionalizedPolymers (C3)

After step 404, step 406 is performed to create a hydrophilic coating ofthe reaction sites surfaces by attachment of hydrophilic polymers asillustrated in FIG. 7.

Coating step 406 is important when a chip is used with an aspect ratioclose to 1, substrates which may leach ions or have other interactionswith the bio-sample/reaction which could lead to inhibition, poisoningof the sample and erroneous or inaccurate results. As mentioned above,according to various embodiments, the goal of coating the chip is toachieve a substantially continuous, hydrophobic film covering substratesurface 106, which will allow for better sample loading and retentionduring reactions and non-interference in the reaction. The reactions maybe during thermal cycling in some embodiments. In some embodiments,next, the glycidyl group made available in the previous step may bereacted with polyethylene glycols, polyvinyl alcohols or polypropyleneglycols of various molecular weights with or without post reaction crosslinking.

One embodiment is illustrated in FIG. 7. After the glycidyl group to thereaction site surfaces in FIG. 6, the glycidyl group is furthermodified. According to various embodiments, there may be two possiblepaths.

In the first possible path, the glycidyl group is reacted withpolyethylene glycols of various molecular weights in presence of basesas catalyst. Di-glycidyl functionalized polyethylene and/orpolypropylene oxides (PEO or PPO) of various molecular weights,represented as C3, are dissolved in a solvent S3. Thus, the glycidylring opens and reacts with the polyethylene glycol —OH. Next, anaddition and reaction with di-glycidyl functionalized polyethyleneand/or polypropylene glycols and subsequent cross linking in presence ofpolyamines generates a continuous film with tunable hydrophilicity.

In a second possible path, the glycidyl group is hydrolyzed. Then, the—OH with di-glycidyl functionalized polyethylene and/or polypropyleneglycols are reacted. This is followed by subsequent cross linking inpresence of polyamines. The reactions are completed via curing in airand oven for given lengths of time and temperatures (room T to 120 C),in one embodiment.

Substrate Surface Second Step: Hydrophobic Silanes (C1))

As mentioned above, step 402 of FIG. 4 covers substrate surface 106 witha hydrophobic layer. However, there is a possibility that there might beuncoated/unreacted parts of surface which may then react during thesubsequent coating steps 404 and 406 of FIG. 4. This may lead tovariation in hydrophobicity of the coating of substrate surfaces 106,which in turn could lead to sample pooling on substrate surface 106while loading. This may lead to sample volumes bridging between reactionsites and erroneous results. To remedy this, the hydrophobic coating ofthe substrate surfaces may be repeated (step 402) using tri-alkoxysilanes functionalized with C4-C20 hydrocarbons to fine tune andhomogenize the hydrophobicity coating of substrate surface 106, in someembodiments.

Next in step 408, the hydrophobic character substrate surfaces of thesubstrate may be fine-tuned. During the previous steps of coating thewalls of reaction sites 104, there may have been some undesiredreactions with the coating on the substrate surface. As such, in thisstep, the hydrophobic coating on the substrate surface may be adjusted.

With reference to FIG. 8, S1 represents solvent 1 (hydrophobic) and S2represents solvent 2 (hydrophilic). S1 and S2 are immiscible solventswith S1 not having access to the area wetted by S2. During step 404,with reference to FIG. 6, some C2 may have been attached to substratesurface 106 reducing the needed/necessary hydrophobicity. In someembodiments, an additional step like step 402 may be implemented againto fine tune the hydrophobicity of the substrate surface 106. This maybe needed to achieve accurate dPCR. The presence of S2 within reactionsites 104 prevent S1 and the alkoxy silane from accessing to thesurfaces of reaction sites 104, maintaining the hydrophilicity.

Step 408 may help to achieve the right balance between the substratesurface hydrophobicity and vertical surface hydrophilicity, and alsobalance with the physical geometric consideration of the substrate, suchas reaction site pitch and thickness of the substrate. This may beimportant in enabling uniform and consistent loading of the reactorswith the bio-samples to be assessed/investigated.

The following is a list of chemicals that may be used to implementembodiments of the present disclosure:

-   -   Type one: silicon derivatives which will allow chemical bonds to        any substrates having “hydrated” SiO2 and subsequently to the        type two chemicals    -   Type two: various (molecular weight and composition) epoxy        polymers to enable cross linking of/with type one chemicals    -   Butyl, hexyl, octyl, decyl, do-decyl, hexadecyl trimethoxy        silane; same C4-C16 hydrocarbons triethoxysilane;        per-fluorinated derivatives of same Cx    -   Aminopropyl trimethoxy and/or triethoxy silane; other amino Cx        trimethoxy and/or triethoxy silanes    -   Glycidylpropyl dimethoxy methylsilane; glycidylpropyl trimethoxy        and/or triethoxysilane; other glycidyl Cx trimethoxy and/or        triethoxy silanes    -   Sodium hydroxide solutions, hydrochloric acid solutions, sodium        hypochlorite solutions    -   Hydrophobic and hydrophilic solvents (pentanes, hexanes,        heptanes, acetonitrile, isopropyl alcohol, ethanol, methanol,        water, ketones, etc)    -   Diglycidyl monomers and/or polymers (polyethylene oxide (any        molecular weight) functionalized diglycidyl, polypropylene oxide        (any molecular weight) functionalized diglycidyl, polyvinyl        alcohol functionalized diglycidyl, small molecular weight Cx        hydrocarbons functionalized diglycidyl, diisocyanates compounds    -   Amines, poly-amines, alcohols, poly-alcohols to be used as        catalysts and cross linking agents

As described above, the chip may be cleaned before coating the chipaccording to various embodiments described herein. Various cleaningprotocols may be implemented. For example, an exemplary cleaningprotocol is as follows:

-   -   1. 1×2 min rinse in IPA; 1×220 kHz; 1×170 kHz    -   2. 1×2 min rinse in ACN; 1×220 kHz; 1×170 kHz    -   3. 4×1 min rinse in DIW; 2×220 kHz; 2×170 kHz    -   4. 30 min in 15% HNO3, 15 min sonic at 220 kHz and 15 min sonic        at 170 kHz    -   5. 1×2 min in 0.14% nitric acid with 1× sonic at 170 kHz and 1×        at 220 kHz

It should be recognized that coating step 402 may be implemented by avariety of protocols.

As an example, the following cleaning protocol, according to embodimentsof the present disclosure, is given:

-   -   1. 45 min in 1.5% ndecyl trimethoxysilane (C10-silane) in        heptanes (substrate surface; vertical surface blocked)    -   2. 15 min in 0.14% nitric acid    -   3. 30 min at RT in air    -   4. 2×2 min in IPA with sonic at 220 kHz 100% power    -   5. 2×2 min in heptanes with sonic at 220 kHz 100% power    -   6. 30 min in 2% 3glycidyloxypropyl trimethoxysilane in heptanes        (all surfaces); 5 min sonic 220 kHz at start    -   7. 2×1 min in ACN with sonic at 220 kHz

It should be recognized that coating step 404 and 406 may be implementedby a variety of protocols. However, as an example, the followingprotocol, according to embodiments of the present disclosure, is given:

-   -   1. 90 min in 2% PEG 8 k and 0.1% TPA in ACN with 5 min sonic at        220 kHz, 100% power    -   2. 30 min in air at RT    -   3. 90 min in oven at 92 C    -   4. 1×2 min in DIW, sonic at 170 kHz, 100% power    -   5. 1×2 min in IPA, sonic at 170 kHz, 100% power    -   6. 1×2 min in heptanes, sonic at 170    -   7. 1×2 min in IPA, sonic at 170 kHz, 100% power    -   8. 1×2 min in aceto nitrile (ACN); sonic at 170 kHz, 100% power    -   9. 2×1 min DIW sonic at 220 kHz    -   10. 2×1 min DIW sonic at 170 kHz    -   11. 15 min in air    -   12. Blow dry with air    -   13. 30 min in oven at 92 C    -   14. React 30 min in 2% 3glycidyloxypropyl trimethoxylsilane in        heptanes; 5 min sonic 220 kHz at start    -   15. 2×1 min in ACN with sonic 1× at 220 kHz and 170 kHz    -   16. React 90 min in 2% Jeff Amine 5 k and 0.1% TPA in ACN with 5        min sonic at 220 kHz, 100% power    -   17. Dry for 15 min in air at RT    -   18. React/dry for 90 min in oven at 92 C    -   19. 1×2 min in IPA, sonic at 170 kHz    -   20. 1×2 min in heptane, sonic at 170 kHz    -   21. 1×2 min in IPA, sonic at 170 kHz    -   22. 1×2 min in aceto nitrile (ACN); sonic at 170 kHz, 100% power    -   23. 2×1 min rinse DIW with sonic 170 kHz    -   24. 1×2 min in 0.5% bleach (5 ml Clorox +95 ml DIW); sonic at        170 kHz    -   25. 4×1 min DIW 2× sonic at 170 kHz and 2× sonic at 220 kHz    -   26. 15 min in air    -   27. Blow dry with air    -   28. 30 min dry in oven at 92 C    -   29. Vacuum pack

Another example of a protocol to implement the coating according toembodiments described herein is described below. The following methodmay work on high aspect ratio chips as well as low aspect ratio chips.For high aspect ratio chips, capillary forces is a more prominent factorin the loading process.

-   -   1. Cleaning; Mikroglas arrays;    -   2. 2×2 min rinse in IPA; 1×220 kHz; 1×170 kHz    -   3. 2×2 min rinse in ACN; 1×220 kHz; 1×170 kHz    -   4. 4×2 min rinse in DIW; 2×220 kHz; 2×170 kHz    -   5. 30 min in 15% HNO3, 15 min sonic at 220 kHz and 15 min sonic        at 170 kHz    -   6. 1×2 min in 0.14% nitric acid with 1× sonic at 170 kHz and 1×        at 220 kHz    -   7. 4×1 min in DIW with sonic at 2×170 kHz and 2×220 kHz    -   8. 2×2 min in IPA with sonic at 220 kHz 100% power    -   9. 2×2 min in heptanes with sonic 1× at 220 kHz and 1× at 170        kHz

A coating method according to various embodiments described herein mayalso meet processing requirement desires. For example, if a substrateused for biological reactions is one that is used by a high volume ofcustomers, the coating method according to various embodiments isreproducible, can meet cost requirements, and scalable to meet demandsfor the use of the substrate.

As mentioned above, an instrument that may be utilized according tovarious embodiments, but is not limited to, is a polymerase chainreaction (PCR) instrument. FIG. 9 is a block diagram that illustrates aPCR instrument 900, upon which embodiments of the present teachings maybe implemented. PCR instrument 900 may include a heated cover 910 thatis placed over a plurality of samples 912 contained in a sample supportdevice (not shown). In various embodiments, a sample support device maybe a glass or plastic slide with a plurality of sample regions, whichsample regions have a cover between the sample regions and heated cover910. Some examples of a sample support device may include, but are notlimited to, a multi-well plate, such as a standard microtiter 96-well, a384-well plate, a chip illustrated in FIG. 1, or a microcard, or asubstantially planar support, such as a glass or plastic slide. Thesample regions in various embodiments of a sample support device mayinclude depressions, indentations, ridges, and combinations thereof,patterned in regular or irregular arrays formed on the surface of thesubstrate. Various embodiments of PCR instruments include a sample block914, elements for heating and cooling 916, a heat exchanger 918, controlsystem 920, and user interface 922. Various embodiments of a thermalblock assembly according to the present teachings comprise components914-918 of PCR instrument 900 of FIG. 9.

For embodiments of PCR instrument 900 in FIG. 9, control system 920, maybe used to control the functions of the detection system, heated cover,and thermal block assembly. Control system 920 may be accessible to anend user through user interface 922 of PCR instrument 900 in FIG. 9.Also a computer system 100, as depicted in FIG. 1, may serve as toprovide the control the function of PCR instrument 900 in FIG. 9, aswell as the user interface function. Additionally, computer system mayprovide data processing, display and report preparation functions. Allsuch instrument control functions may be dedicated locally to the PCRinstrument, or a computer system may provide remote control of part orall of the control, analysis, and reporting functions.

The following descriptions of various implementations of the presentteachings have been presented for purposes of illustration anddescription. It is not exhaustive and does not limit the presentteachings to the precise form disclosed. Modifications and variationsare possible in light of the above teachings or may be acquired frompracticing of the present teachings. Additionally, the describedimplementation includes software but the present teachings may beimplemented as a combination of hardware and software or in hardwarealone. The present teachings may be implemented with bothobject-oriented and non-object-oriented programming systems.

Exemplary systems for methods related to the various embodimentsdescribed in this document include those described in following U.S.provisional patent applications:

-   -   U.S. provisional application No. 61/612,087, filed on Mar. 16,        2012; and    -   U.S. provisional application No. 61/723,759, filed on Nov. 7,        2012; and    -   U.S. provisional application No. 61/612,005, filed on Mar. 16,        2012; and    -   U.S. provisional application No. 61/612,008, filed on Mar. 16,        2012; and    -   U.S. provisional application No. 61/723,658, filed on Nov. 7,        2012; and    -   U.S. provisional application No. 61/723,738, filed on Nov. 7,        2012; and    -   U.S. provisional application No. 61/659,029, filed on Jun. 13,        2012; and    -   U.S. provisional application No. 61/723,710, filed on Nov. 7,        2012; and    -   U.S. provisional application No. 61/774,499, filed on Mar. 7,        2013; and    -   PCT International Application No. PCT/US2013/032002, filed Mar.        15, 2013; and    -   PCT International Application No. PCT/US2013/032420, filed Mar.        15, 2013; and    -   PCT International Application No. PCT/US2013/032107, filed Mar.        15, 2013; and    -   PCT International Application No. PCT/US2013/032242, filed Mar.        15, 2013; and    -   PCT International Application No. PCT/US2013/031890, filed Mar.        15, 2013.

All of these applications are also incorporated herein in their entiretyby reference.

Although various embodiments have been described with respect to certainexemplary embodiments, examples, and applications, it will be apparentto those skilled in the art that various modifications and changes maybe made without departing from the present teachings.

What is claimed is:
 1. An apparatus for biological reactions, theapparatus comprising: a substrate comprising a substrate surface; and aplurality of reaction sites disposed along the substrate surface, eachreaction site spatially separated from the remaining reaction sites,each reaction site comprising a reaction site surface; a coatingmaterial, wherein: the coating material coats the substrate surface, toprovide a coated substrate surface; the coating material coats each ofthe reaction site surfaces to provide a plurality of coated reactionsites; the coated substrate surface and the coated reaction sitesurfaces are hydrophilic; the coated substrate surface has a watercontact angle of 60-90 degrees; and the coated substrate surface has anadvancing contact angle of 70-85 degrees; wherein each of the coatedreaction site surfaces is configured to load the reaction sites with asample; and wherein each sample is configured to undergo a biologicalreaction within the reaction site.
 2. The apparatus of claim 1, whereinthe coating material is configured to prevent pooling of a sample on thesubstrate surface by providing substrate surface with an advancingcontact angle of 70 to 85 degrees and with a hysteresis of less than 30degrees.
 3. The apparatus of claim wherein the hysteresis of thesubstrate surface is zero degrees or is between 0-15 degrees.
 4. Theapparatus of claim 1, wherein the reaction sites are configured to holdat most 1 nanoliter of a liquid sample.
 5. The apparatus of claim 1,wherein the plurality of reaction sites are through-holes.
 6. Theapparatus of claim 1, wherein the hydrophilicities of the coatedsurfaces are configured to retain amplification reagents in theplurality of reaction sites.
 7. The apparatus of claim 1, whereinhydrophilicities of the coated surfaces generate a sufficient surfacetension force to substantially confine the sample within each loadedreaction site.
 8. The apparatus of claim 1, wherein a dimension of eachreaction site is configured to provide an amount of capillary action,wherein the capillary action determines a volume of liquid sample loadedinto each reaction site.
 9. The apparatus of claim 1, further comprisinga loader configured to receive the sample and to load the sample into atleast some of the reaction sites, wherein the coating material isconfigured to reduce or prevent pooling of a sample on the substratesurface such that all of the sample is disposed within the reactionsites and none of the sample is contained on the surface.
 10. Theapparatus of claim 1, wherein a hydrophilicities of the coated reactionsite surfaces is the same as a hydrophilicity of coated substratesurface.
 11. The apparatus of claim 1, wherein the substrate comprisesat least one of silicon, a silicon oxide material, a glass material, ametal material, or a ceramic material.
 12. The apparatus of claim 1,wherein the substrate comprises a silicon oxide material.
 13. Anapparatus for biological reactions, the apparatus comprising: a loaderconfigured to receive a sample; a substrate comprising a substratesurface; and a plurality of reaction sites disposed along the substratesurface, each reaction site spatially separated from the remainingreaction sites, each reaction site comprising a reaction site surface; acoating material, wherein: the coating material coats the substratesurface to provide a coated substrate surface; the coating materialcoats each of the reaction site surfaces to provide a plurality ofcoated reaction sites; the coated substrate surface and the coatedreaction site surfaces are hydrophilic; the coated substrate surface hasa water contact angle of 60-90 degrees:, the coated substrate surfacehas an advancing contact angle of 70-85 degrees; and the substratesurface has a hysteresis of less than 30 degrees; wherein the loader isconfigured to contact the substrate surface to load the sample into atleast some of the reaction sites of the substrate such that all of thesample is disposed within the reaction sites and none of the sample iscontained on the substrate surface; and wherein each sample isconfigured to undergo a biological reaction within the reaction site.14. The apparatus of claim 13, wherein the coating material has anadvancing contact angle of 70 to 85 degrees so as to reduce or preventpooling of a sample on the substrate surface such that all of the sampleis disposed within the reaction sites and none of the sample iscontained on the surface.
 15. The apparatus of claim 13, wherein thesubstrate comprises at least one of silicon, a silicon oxide material, aglass material, a metal material, or a ceramic material.
 16. Theapparatus of claim 13, wherein the substrate comprises a silicon oxidematerial.
 17. An apparatus for biological reactions, the apparatuscomprising: a substrate comprising a substrate surface; and a pluralityof reaction sites disposed along the substrate surface, each reactionsite spatially separated from the remaining reaction sites, eachreaction site comprising a reaction site surface; a coating material,wherein: the coating material coats the substrate surface to provide acoated substrate surface: the coating material coats each of thereaction site surfaces to provide a plurality of coated reaction sites;the coated substrate surface and the coated reaction site suffixes arehydrophilic; the coated substrate surface has a water contact angle of60-90 degrees; the coated substrate surface is characterized by anadvancing contact angle of 70-85 degrees; and wherein the coatedsubstrate surface is characterized by a hysteresis of less than 30degrees; wherein each of the coated reaction site surfaces is configuredto load the reaction sites with a sample; and wherein each sample isconfigured to undergo a biological reaction within the reaction site.18. The apparatus of claim 17, wherein the hysteresis of the substratesurface is zero degrees or is between 0-15 degrees.
 19. The apparatus ofclaim 17, wherein the substrate comprises at least one of silicon, asilicon oxide material, a glass material, a metal material, or a ceramicmaterial.
 20. The apparatus of claim 17, wherein the substrate comprisesa silicon oxide material.